A Physiological Function of Inflammation-Associated SerpinB2 Is Regulation of Adaptive Immunity This information is current as Wayne A. Schroder, Thuy T. T. Le, Lee Major, Shayna of September 26, 2021. Street, Joy Gardner, Eleanore Lambley, Kate Markey, Kelli P. MacDonald, Richard J. Fish, Ranjeny Thomas and Andreas Suhrbier J Immunol 2010; 184:2663-2670; Prepublished online 3 February 2010; Downloaded from doi: 10.4049/jimmunol.0902187 http://www.jimmunol.org/content/184/5/2663 Supplementary http://www.jimmunol.org/content/suppl/2010/02/01/jimmunol.090218 http://www.jimmunol.org/ Material 7.DC1 References This article cites 68 articles, 21 of which you can access for free at: http://www.jimmunol.org/content/184/5/2663.full#ref-list-1 Why The JI? Submit online. by guest on September 26, 2021 • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2010 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology A Physiological Function of Inflammation-Associated SerpinB2 Is Regulation of Adaptive Immunity Wayne A. Schroder,* Thuy T. T. Le,* Lee Major,* Shayna Street,† Joy Gardner,* Eleanore Lambley,* Kate Markey,* Kelli P. MacDonald,* Richard J. Fish,‡ Ranjeny Thomas,† and Andreas Suhrbier* SerpinB2 (plasminogen activator inhibitor-2) is widely described as an inhibitor of urokinase plasminogen activator; however, SerpinB22/2 mice show no detectable increase in urokinase plasminogen activator activity. In this study, we describe an unexpected immune phenotype in SerpinB22/2 mice. After immunization with OVA in CFA, SerpinB22/2 mice made 6-fold more IgG2c and generated 2.5-fold more OVA-specific IFN-g–secreting T cells than SerpinB2+/+ littermate controls. In SerpinB2+/+ mice, high inducible SerpinB2 expression was seen at the injection site and in macrophages low levels in draining lymph nodes and conventional dendritic cells, and no expression was seen in plasmacytoid dendritic, B, T, or NK cells. SerpinB22/2 macrophages promoted greater Downloaded from IFN-g secretion from wild-type T cells in vivo and in vitro and, when stimulated with anti-CD40/IFN-g or cultured with wild-type T cells invitro, secreted more Th1-promoting cytokines than macrophages from littermate controls. Draining lymph node SerpinB22/2 myeloid APCs similarly secreted more Th1-promoting cytokines when cocultured with wild-type T cells. Regulation of Th1 responses thus appears to be a physiological function of inflammation-associated SerpinB2; an observation that may shed light on human inflammatory diseases like pre-eclampsia, lupus, asthma, scleroderma, and periodontitis, which are associated with SerpinB2 poly- morphisms or dysregulated SerpinB2 expression. The Journal of Immunology, 2010, 184: 2663–2670. http://www.jimmunol.org/ erpinB2 is a member of the Clade B or OVA-like serine shown to inhibit uPA in vitro (3, 4). The majority of the .860 pub- protease inhibitor (ov-serpin) subgroup of the serpin su- lications listed in PubMed on SerpinB2/PAI-2 have thus assumed that S perfamily, which includes proteinase inhibitors 6, 8, and 9, the principle role of this serpinis inhibition of uPA,although a number MENT, Bomapin, and maspin. Ov-serpins lack a classical secretory of reports have indicated that certain activities associated with Ser- signal peptide and are frequently found to have cytoplasmic or nu- pinB2 expression appear unrelated to uPA inhibition (2, 6, 9, 11–14). cleocytoplasmic distributions and functions (1, 2). SerpinB2 is in- Although SerpinB2 can inhibit uPA in vitro, the evidence that this duced during many inflammatory processes and infections (3–7) and represents a physiological function for SerpinB2 in vivo is not com- by guest on September 26, 2021 is one of the most upregulated proteins of activated monocytes/mac- pelling. SerpinB22/2 mice show no apparent defects in uPA activity, rophages representing up to 1% of total protein (7, 8). SerpinB2 can and mice deficient in both SerpinB2 and PAI-1 show no additional also be induced to a lesser extent in fibroblasts and endothelial cells defects in uPA activity over PAI-12/2 mice (15). Furthermore, co- and is constitutively expressed by differentiating keratinocytes, pla- valent uPA-SerpinB2 complexes, which are easily generated in vitro, cental trophoblasts (3), adipocytes (9), and a range of tumors (4, 10). have not been unequivocally demonstrated invivo, whereas uPA-PAI- SerpinB2 is also known as plasminogen activator inhibitor (PAI) 1 complexes have been readily detected (16). The majority of Ser- type 2 and is widely described as an inhibitor of the extracellular pinB2 is also retained intracellularly, because it is inefficiently se- urokinase plasminogen activator (uPA), because SerpinB2 can be creted (3). The physiological function of inflammation-associated SerpinB2 thus remains unclear (4). Clinically, dysregulated SerpinB2 expression or SerpinB2 poly- *Queensland Institute of Medical Research, Australian Center for International and morphisms have been associated with a number of diseases involving Tropical Health, Griffith Medical Research College; †Diamantina Institute for Can- cer, Immunology and Metabolic Medicine, University of Queensland, Princess Alex- inflammation. Decreased expression is associated with pre-eclampsia andra Hospital, Brisbane, Queensland, Australia; and ‡Department of Genetic (17), whereas expression is upregulated in asthma (18), periodontal Medicine and Development, Faculty of Medicine, University of Geneva, Geneva, disease (19), and hyperkeratotic corn tissue (20). Increased SerpinB2 Switzerland expression in certain cancers is also associated with improved prog- Received for publication July 7, 2009. Accepted for publication December 8, 2009. nosis (10). SerpinB2 polymorphisms have also been associated with This work was supported by grants from the National Health and Medical Research antiphospholipid syndrome (21), lupus (22), and myocardial in- Council of Australia and the National Cancer Institute (R01 CA098369). A.S. is a principal research fellow of the National Health and Medical Research Council. farction in some (23) but not other studies (24). SerpinB2 has been Address correspondence and reprint requests to Dr. Andreas Suhrbier, Queensland reported to have a bewildering array of activities including regulation Institute of Medical Research, Post Office Royal Brisbane Hospital, Queensland of monocyte and keratinocyte proliferation and differentiation (13, 4029, Australia. E mail address: [email protected] 25, 26), inhibition of apoptosis in some (5, 14, 27) but not other set- The online version of this article contains supplemental material. tings (28), inhibition of necrosis (29), inhibition of the IL-1b con- Abbreviations used in this paper: BMDM, bone marrow-derived macrophage; cDC, verting enzyme (30), inhibition of retinoblastoma protein degradation conventional DC dendritic cell; DC, dendritic cell; ov-serpin, OVA-like serine pro- tease inhibitor; PAI, plasminogen activator inhibitor; pDC, plasmacytoid dendritic (31), and priming of IFN-a/b responses (6). SerpinB2 has been re- cell; poly(I:C), polyinosinic:polycytidylic acid; RPM, resident peritoneal macro- ported to bind annexins (32), the retinoblastoma proteins (2, 12), IFN phage; TEPM, thioglycollate-elicited peritoneal macrophage; uPA, urokinase plas- response factor 3 (33), ZNF198/FGFR1 fusion kinase (34), protea- minogen activator. some subunit, b type 1 (35), and vitronectin (36). These numerous Copyright Ó 2010 by The American Association of Immunologists, Inc. 0022-1767/10/$16.00 activities and the limited evidence supporting a role in uPA inhibition www.jimmunol.org/cgi/doi/10.4049/jimmunol.0902187 2664 SerpinB2 REGULATES Th1 RESPONSES leave no clear basis for understanding the clinical observations as- without 100 ng/ml LPS (Escherichia coli O55:B5; Sigma-Aldrich). Su- sociated with SerpinB2. pernatants were concentrated 10-fold using Microcon centrifugal filter unit Using SerpinB22/2 mice (15), we sought to gain insights into with a 3000 m.w. cutoff (Millipore, North Ryde, New South Wales, Australia), and uPA activity was measured using the Mouse uPA activity the physiological role of inflammation-associated SerpinB2. Only 2 2 assay kit (Innovative Research, Novi, MI). For proliferation assays, sple- two phenotypes have so far been reported for SerpinB2 / mice: nocytes from SerpinB22/2 and SerpinB2+/+ mice were incubated in du- increased susceptibility to multistage skin carcinogenesis (31) and plicate with 0.5 mg/ml anti-CD3 and/or 100 ng/ml LPS for 2 d, and increased adipocyte hypotrophy after a high-fat diet (9). Neither proliferation was measured by MTS assay (Promega, Madison, WI). phenotype provides discernable insights into the clinical ob- FACS analysis 2/2 servations. In this study, using SerpinB2 mice that have been FACS analysis was undertaken